NEAT1 variant 1 weakens the genome-wide effect of miR-3122 on blocking H3K79me3 in bladder cancer

Nuclear-enriched abundant transcript 1 (NEAT1) is one of the most well-studied long non-coding RNAs (lncRNAs) in multiple human carcinoma. Two distinct variants of NEAT1, however, are never illuminated their specific functions and mechanisms underlying carcinogenesis. In this study, biotin-labelled NEAT1 variants were generated to incubate with cell lysate of bladder cancer cell T24 cells, and fished a batch of RNA substances. Here, we observed that NEAT1.1 (the short transcript) could capture 122 microRNAs (miRNAs), 36 small nucleolar RNAs (snoRNAs), 55 lncRNAs and 38 mRNAs while NEAT1.2 (the long transcript) could obtain 142 miRNAs, 51 snoRNAs, 72 lncRNAs and 41 mRNAs. Furthermore, we also found that the distinctions of RNA binding substances between these two variants were mainly expressed in nucleus rather than cytoplasm. GO analysis indicated that these non-coding RNAs governed histone modification, nucleosome assembly and chromosome organization. We picked up miRNA miR-3122, which substantially interacted with NEAT1.1, and found that histone H3K79me3 was reduced in bladder cancer T24, BIU-87 and EJ-1 cells after miR-3122 overexpression, and rescued by NEAT1.1 additional compensation. Nonetheless, we failed to find that miR-3122 could interfere with expression of H3K79 methyltransferase disruptor of telomeric silencing-1 like (DOT1L). Interestingly, we harvested histone 3 fished by biotin-labelled miR-3122, and validated this intercrossing using RNA immunoprecipitation. Taken together, we demonstrated that NEAT1.1 weakened the effect of miR-3122 on H3K79me3 suppression in bladder cancer.


INTRODUCTION
Bladder cancer (BC), one of the most common urogenital tumors, presents high incidence, prevalence, recurrence and mortality [1,2]. The multiple factors are involved in etiology of BC, including genetic, epigenetic and environmental factors. The conventional therapies of BC start with surgical resection followed by adjuvant therapies, such as radiotherapy and chemotherapy. The adjuvant therapies are introduced in particular when the tumor appears loco-regional and distant invasion. Effective chemotherapeutic and targeted drugs were introduced to systematically advance the treatment options in BC over the last two decades. However, the tumor resistance to therapy is still a major challenge in individual treatment with low toxicity and significant benefit. Tumor can become tolerant to these pharmaceutical treatments including resistance emerges as well as early tumor recurrence.
Emerging evidence shows that long non-coding RNAs (lncRNAs) have attracted more and more attention as one of the epigenetic regulatory factors and potential therapeutic targets for multiple cancers [3]. One lncRNA named nuclear paraspeckle assembly transcript 1 (NEAT1) has been widely acknowledged to play a crucial role in malignancy on BC and other diseases [4][5][6]. NEAT1 participates in multiple biological pathways through a diverse group of mechanisms due to the formation of the paraspeckle, which it can influence the stability of a tumor to develop chemoresistance [7]. Interestingly, two existing variants of NEAT1 serve the opposite effects on the capabilities of tumor growth, apoptosis and invasion. Knockdown of NEAT1.1 (the short transcript) in vitro could inhibit cell invasion and proliferation [8], while knockdown of NEAT1.2 (the long transcript) promoted cell growth [9]. One explanation is the architectural functions in the construction of the nuclear body [10]. However, the complicated regulatory network of different NEAT1 variants for BC tumorigenesis is not fully understood.
In this study, we produced full length of NEAT1.1 and 1.2 with biotin tag, and fished the interacted RNAs. We analyzed them and finally focused on the epigenetic function of miR-3122 in BC cells. Our research reported a novel regulatory mechanism of NEAT1 in BC tumorigenesis, and provided an example of epigenetic study on other lncRNAs expressed in nucleus.  Figure 1A). In this list of differential binding RNAs, nearly all of them were expressed in nucleus and chromatin ( Figure 1B) according to database of "RNALOCATE" [11]. Furthermore, we also found that the distinctions of RNA binding substances between these two variants were mainly expressed in nucleus rather than cytoplasm. In summary, we characterized the binding RNA profiles of NEAT1 in BC T24 cells.

Binding RNA profiling of NEAT1 in BC cells
The intercrossing between miR-3122 and NEAT1.1 Next, we picked up miR-3122, which had the most significant in differential binding RNAs (p = 3.4x10 -8 ).
Our FISH assay validated that miR-3122 and NEAT1.1 had a substantial interaction with each other instead of NEAT1.2 ( Figure 2A). Furthermore, we also determined that miR-3122 could bind with 1901 to 2800 of NEAT1.1 ( Figure 2B-2E) as we prepared the different truncation of NEAT1.1 ( Figure 2F). Taken together, we demonstrated the intercrossing between miR-3122 and NEAT1.1.

Mono-methylation of H3K79 inhibited by miR-3122
We had found that miR-3122 was mainly expressed in nucleus, it did not look like regular studies looking for mRNA targets in the cytoplasm. To investigate the epigenetic role of miR-3122 in nucleus of BC cells, we performed dot plot assay to study the histone modifications. From the pattern of histone H3 methylation, we unexpectedly observed that histone H3K79me3 was significantly reduced after miR-3122 overexpression, and rescued by NEAT1.1 additional compensation in T24 cells ( Figure 3A), which was further verified in BIU-87 and EJ-1 cells ( Figure 3B, 3C). Nonetheless, we failed to find that miR-3122 could interfere with expression of H3K79 methyltransferase disruptor of telomeric silencing-1 like (DOT1L) ( Figure 3D-3F).
Similarly with molecular fishing of NEAT1, biotinlabelled miR-3122 was used to search the binding protein by mass spectrum in T24 cells, and we harvested histone H3 as one of the top plausible binding proteins with K79 trimethylation ( Figure 4A). Consistently, RNA immunoprecipitation assay was conducted to validate the intercrossing between miR-3122 and histone H3 in T24, BIU-87 and EJ-1 cells. We found that H3K79me3 could obtain the highest enrichment of miR-3122 compared to other H3 histone modifications ( Figure 4B), indicating that H3K79me3 was attributed to miR-3122. Overall, we determined that miR-3122 would affect genome-wide H3K79me3 to regulate gene transcription.

DISCUSSION
NEAT1 is a well-acknowledged lncRNA functioning in development and diseases via affecting multiple signaling pathways. In the current massive literature, NEAT1 is entangled with various miRNAs in cytoplasm, even exosome. However, both of two NEAT1 variants are actually expressed in nucleus. NEAT1.2 contributes to building phase-separated nuclear paraspeckles via its high-affinity binding sites for recruitment of the essential paraspeckle proteins (NONO and SFPQ) [10,12]. However, NEAT1.1 containing with only two domains for RNA stability and isoform selection seems to have contributed less to paraspeckle formation. The mechanism behind the opposite effects on tumor of NEAT1.1 and NEAT1.2 still remain obscure [13]. According to previous studies of the same kind non-coding RNAs in nucleus [14,15], lncRNA could facilitate the connection between transcription factors or epigenetic regulators and DNA elements. In our research, our data unexpectedly supports that a large number of nucleus-expressed RNAs are enriched on NEAT1. Unlike the cytoplasmexpressed miRNA such as miR129-5p [16], miR214 [17], miR342-3p [18], miR141-3p [19], most of them especially in nucleus are less studied. We have no clue about the binding RNAs in other cancer, but the role of NEAT1 in BC needs to be re-examined.
The different binding RNAs between NEAT1 variants suggest that they NEAT1.1 has a distinct spatial   structure from NEAT1.2 although the sequence of NEAT1.1 overlaps with the part of NEAT1.2. Taking miR-3122 in this study as an example, it can be shown that the miR-3122 binding domain of NEAT1.1 may be exposed to surface compared to NEAT1.2, indicating that lncRNAs served as sponging to miRNAs may be attributed to their alternative 2D even 3D structures.
So far, miRNA binding proteins are majorly centered around the argonaute (AGO)-family proteins-mediated miRNA-induced silencing complex [20,21]. Emerging evidence indicating that other (RBPs) such as AUF1 and HuR can also directly interact with mature miRNAs [22,23] also implicates the competitive binding of RBPs to target miRNAs, sequestration of miRNAs from AGO, promotion of AGO binding to miRNAs, and transfer of miRNAs from RBPs to AGO [24]. Although our data determines the coincidence of miR-3122 enrichment and H3K79me3 variation, we speculate that miR-3122 is supposed to bind with genomic DNA rather than histone 3 directly, and the binding regions packaged with nucleosome and controlling the transcription activity of target genes might have some unknown factors to recruit miR-3122. More experiments are needed to confirm this hypothesis in future study.
We summarize the binding RNAs of NEAT1.1 in BC cells, and find that NEAT1.1 can weaken the miR-3122-mediated suppressive effect on genome-wide H3K79me3. Our observations provide a brand-new action mode of lncRNA-miRNA for epigenetic regulation in cancer.

Cell culture
Bladder cancer cell T24, BIU-87 and EJ-1 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Beijing, China) were cultured in 1640 medium with 10% FBS (HyClone, USA) at the incubator with 37° C, 5% CO2 and 100% humidity.

RNA synthesis in vivo
Full or truncated single-strand RNA products of NEAT1.1 and miR-3122 labelled by biotin were synthetized by Genepharma (Shanghai, China). Full length of NEAT1.2 was initially amplified by PCR using forward primer adding T7 promoter sequence. PCR products were purified as a template for RNA synthesis using T7 RNA polymerase kit (Cat. No. M0251L, NEB, USA). In the reaction system preparation, uridine nucleoside triphosphate (UTP) was replaced by the specific biotin labelled UTP (Cat. No. AM8451, Thermo Fisher Scientific, USA). DNase I (Cat. No. M0303S, NEB) was used for template removal, followed by ethanol precipitation. RNA products were examined the concentration and subpackaged to store at -80° C for the subsequent experiments.
1×10 7 cells were treated by 200 μL cell lysis buffer for 30 min on ice with the gently and repeatedly pipetteblowing, and added the freshly prepared RNA-beads mixture for 2-hrs gentle rotation at 4° C. After that, beads were washed by 400 μL cell lysis buffer for five times. For next generation sequence, beads were resuspended by 1 mL Trizol for RNA isolation. The final purified RNA was used for library construction. For mass spectrum, beads were resuspended by 1x protein loading buffer. Proteins were processed under metal bath at 100° C for 10 min, and the supernatant was transferred to a new tube without beads. Mass spectrum service is provided by Shanghai OEbiotech (Shanghai, China).

Dot plot
1×10 7 cells were treated by 400 μL cell lysis buffer containing 1x protease and phosphatase inhibitor cocktail for 30 min on ice with the gently and repeatedly pipette-blowing, then centrifuged with 14000 rpm for 15 min at 4° C. The total protein supernatant was determined the concentration, and dropped 2 μl each on nitrocellulose membrane with 2fold serially dilution (0, 5, 10 and 20 μg) for dot blot assay. The membrane was dried and incubated with H3K4me3 (Cat. No. 61379, Activemotif, USA), H3K9me3 (Cat. No. 39285, Activemotif), H3K27me3 (Cat. No. 61017, Activemotif), H3K79me3 (Cat. No. NB21-1383, Novus, USA) for 4° C overnight with gentle shaking. The membranes were washed by TBST three times at room temperature, followed by rabbit anti-mouse or donkey anti-rabbit IgG-HRP (1:10000 dilution, Beyotime Biotechnology, China) in 10 ml of TBST for 1 h at room temperature with gentle shaking, and washed again for three times. After that AGING membranes were incubated with 3 ml of ECL Western Blotting Substrate (Beyotime Biotechnology, China) for 5 min in darkness at room temperature and developed the spots.

Western blot assay
1×10 7 cells were treated by 400 μL cell lysis buffer containing 1x protease and phosphatase inhibitor cocktail for 30 min on ice with the gently and repeatedly pipette-blowing, then centrifuged with 14000 rpm for 15 min at 4° C. The total protein supernatant was determined the concentration. 40 μg total protein for each sample was added 1x protein loading buffer, and processed under metal bath at 100° C for 10 min, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene fluoride membranes. The membranes were then blocked by 5% non-fat milk TBS for 90 min and then incubated with the respective primary antibodies of DTO1L (1:2000, Cat. No. NB100-40845, Novus) and β-Actin (1:5000, Cat. No. AF5001, Beyotime Biotechnology) overnight at 4° C. Membranes were washed in TBST and incubated with rabbit anti-mouse and goat anti-rabbit IgG-HRP at room temperature for 1 h. After that membranes were incubated with 3 ml of ECL Western Blotting Substrate for 5 min in darkness at room temperature and developed the bands.

Fluorescence in situ hybridization assay
As previously described [33], the FISH probes for NEAT1.1 labelled with R-Phycoerythrin (excitation wavelength 565 nm), for miR-3122 labelled with FITC (excitation wavelength 495 nm) were purchased from G&P Medical Enterprises (Beijing, China). Slides filled with T24 cells washed by PBS were hypotension treatment in 0.075 M KCl at 37° C for 25 min, fixed by pure ethanol for 10 min and dropped on the glass slide, then aged at 56° C for 60 min. The slides were washed by PBS twice and dehydrated with the 70%, 85% and 100% ethanol for 3 min in order, and naturally dried. 10 μl probes were added on and mounted immediately. Slides were next denatured 75° C for 5 min and 42° C for 16 h, then removed the cover glass and incubated in 2 × SSC for 5 min and 0.1% NP-40/2 × SSC for another 2 min at 46° C, followed by 70% ethanol at room temperature for 3 min and dried in dark. 15 μl DAPI were dropped and mounted again. After 10 min incubation in dark, the slides were observed under the fluorescence microscope with the appropriate filters.

Statistical analysis
All experimental data were processed and analyzed using SPSS 22.0 statistical software. The measurement data were expressed as mean ± standard deviation. Oneway ANOVA was used for comparison between multiple groups. The p-value less than 0.05 was considered as statistical significance.

AUTHOR CONTRIBUTIONS
Z.W. performed all experiments and harvested and analyzed the raw data; S.F. and Z.L. assisted experiments design and data analysis; O.J. designed and supported the entire project, as well as drafted and revised manuscript.

CONFLICTS OF INTEREST
The Authors declare that they have no conflicts of interest.

FUNDING
This research was supported by Suzhou Science and technology projects (Grant No. SLC201906).